Nimesulide inhibits proliferation and induces apoptosis of pancreatic cancer cells by enhancing expression of PTEN

Abstract

Pancreatic cancer is the fourth leading cause of cancer-associated cases of mortality worldwide. Prostaglandin-endoperoxide synthase 2 (COX-2) is considered a therapeutic target for prevention of pancreatic cancer. Nimesulide, a selective COX-2 inhibitor, can induce cell apoptosis, resulting in an anti-cancer effect. However, the mechanism underlying this effect remains to be elucidated. The present study aimed to evaluate the effects of nimesulide on proliferation of PANC-1 cells using an MTT assay. Apoptosis was evaluated by DNA laddering and Annexin V-fluorescein isothiocyanate/propidium iodide-stained flow cytometry. Furthermore, western blot analysis was used to elucidate the mechanism underlying nimesulide treatment in PANC-1 cells. It was determined that proliferation of PANC-1 cells was inhibited by nimesulide in a dose-dependent manner. Nimesulide promoted apoptosis of PANC-1 cells. Western blot analysis demonstrated that nimesulide increased expression of cleaved caspase-3 and apoptosis regulator Bax (Bcl-2 associated protein X), and decreased the expression of pro-caspase-3 and apoptosis regulator Bcl-2 (B-cell lymphoma 2). Furthermore, nimesulide enhanced expression of phosphatase and tensin homolog (PTEN), and decreased the expression level of COX-2 and vascular endothelial growth factor. In summary, the results of the present study demonstrated that nimesulide could induce apoptosis and inhibit growth of PANC-1 cells by enhancing the expression of PTEN, which indicates the potential of nimesulide to prevent tumor angiogenesis.

Keywords: apoptosis; nimesulide; pancreatic cancer cells; phosphatase and tensin homolog; proliferation; prostaglandin-endoperoxide synthase 2.

Figure 1.

Apoptosis analysis of PANC-1 cells following treatment with nimesulide. (A) DNA laddering revealed the presence of apoptotic DNA fragmentation following treatment with different concentrations of nimesulide (0, 25, 50, 100, 200 and 400 µmol/l) for 48 h. PANC-1 cells were treated with nimesulide at a concentration of (B) 0, (C) 50, (D) 100, (E) 200 and (F) 400 µmol/l for 48 h and the apoptosis rate was determined by flow cytometry with Annexin V-fluorescein isothiocyanate/propidium iodide double dye assay. (G) Flow cytometry results were analyzed quantitatively. Data are presented as the mean ± standard deviation (n=3). *P<0.05 and **P<0.01 vs. the control group.

Figure 2.

Effects of nimesulide on expression of cleaved-caspase-3, pro-caspase-3, Bax and Bcl-2 in PANC-1 cells. (A) PANC-1 cells were treated with different concentrations of nimesulide (0, 25, 50, 100, 200 and 400 µmol/l) for 48 h and protein expression was determined by western blotting. Expression of (B) cleaved caspase-3, (C) pro-caspase-3, (D) Bax and (E) Bcl-2 was analyzed. *P<0.05 and ***P<0.001 vs. the control group. Bax, Bcl-2 associated protein X; Bcl-2, B-cell lymphoma 2.

Figure 3.

Effects of nimesulide on expression of COX-2 in PANC-1 cells. PANC-1 cells were treated with different concentrations of nimesulide (0, 25, 50, 100, 200 and 400 µmol/l) for 48 h and COX-2 expression was evaluated using western blot analysis. *P<0.05 and **P<0.01 vs. the control group. COX-2, prostaglandin-endoperoxide synthase 2.

Figure 4.

Effects of nimesulide on the expression of PTEN and VEGF in PANC-1 cells. PANC-1 cells were treated with different concentrations of nimesulide (0, 25, 50, 100, 200 and 400 µmol/l) for 48 h and expression of PTEN and VEGF was evaluated using western blot analysis. Treatment with nimesulide increased the expression of PTEN and decreased the expression of VEGF. *P<0.05 and **P<0.01 vs. the control group. PTEN, phosphatase and tensin homolog; VEGF, vascular endothelial growth factor.